Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia.

Dendritic cells (DCs) use all-trans retinoic acid (ATRA) to promote characteristic intestinal responses, including Foxp3(+) Treg conversion, lymphocyte gut homing molecule expression, and IgA production. How this ability to generate ATRA is conferred to DCs in vivo remains largely unstudied. Here, we observed that among DCs, retinaldehyde dehydrogenase (ALDH1), which catalyzes the conversion of retinal to ATRA, was preferentially expressed by small intestine CD103(+) lamina propria (LP) DCs. Retinoids induced LP CD103(+) DCs to generate ATRA via ALDH1 activity. Either biliary or dietary retinoids were required to confer ALDH activity to LP DCs in vivo. Cellular retinol-binding protein II (CRBPII), a cytosolic retinoid chaperone that directs enterocyte retinol and retinal metabolism but is redundant to maintain serum retinol, was required to confer ALDH activity to CD103(+) LP DCs. CRBPII expression was restricted to small intestine epithelial cells, and ALDH activity in CRBPII(-/-) DCs was restored by transfer to a wild-type recipient. CD103(+) LP DCs from CRBPII(-/-) mice had a decreased capacity to promote IgA production. Moreover, CD103(+) DCs preferentially associated with the small intestine epithelium and LP CD103(+) DC ALDH activity, and the ability to promote IgA production was reduced in mice with impaired DC-epithelia associations. These findings demonstrate in vivo roles for the expression of epithelial CRBPII and lumenal retinoids to imprint local gut DCs with an intestinal phenotype.

software to determine the nerve volume density of PGP9.5 positive cell bodies and nerve fibers as a percentage of total tissue area (μm2).The nerve volume densities were significantly decreased in the jejunum (23.7 vs. 31.3;p < 0.001), ileum (29.6 vs. 34.5;p < 0.001) and colon (31.8 vs. 34.3;p < 0.05) of GF as compared to SPF mice.Similar to our observations, there were no significant differences between the nerve volume densities of the germ-free and control stomach (33.5 vs. 33.4;p > 0.05) and duodenum (30.1 vs. 28.8;p > 0.05).These findings suggest that endogenous microbiota are necessary for the normal patterning of the ENS in the early postnatal period, particularly in the mid to distal small intestine and colon.Further studies are needed to determine whether the mechanisms by which the endogenous microbiota act on the ENS involve a direct effect on neural cells.

BACKGROUND:Indoleamine 2,3 dioxygenase-1 (IDO1) is a tryptophan catabolizing enzyme with immunotolerance-promoting functions. We sought to determine if increased gut expression of IDO1 in Crohn's disease (CD) would result in detectable changes in serum levels of tryptophan and the initial IDO1 pathway catabolite, kynurenine. METHODS:Individuals were prospectively enrolled through the Washington University Digestive Diseases Research Center. The Montreal Classification was used for disease phenotyping. Disease severity was categorized by the Physician's Global Assessment. Serum tryptophan and kynurenine were measured by high-pressure liquid chromatography. IDO1 immunohistochemical staining was performed on formalin-fixed tissue blocks. RESULTS:In all, 25 CD patients and 11 controls were enrolled. Eight CD patients had serum collected at two different timepoints and levels of disease activity compared. Strong IDO1 expression exists in both the lamina propria and epithelium during active CD compared to controls. Suppressed serum tryptophan levels and an elevated kynurenine/tryptophan (K/T) ratio were found in individuals with active CD as compared to those in remission or the control population. K/T ratios correlated positively with disease activity as well as with C-reactive protein and erythrocyte sedimentation rate. In the subgroup of CD patients with two serum measurements, tryptophan levels were elevated while kynurenine levels and the K/T ratio lowered as the disease activity lessened. CONCLUSIONS:IDO1 expression in CD is associated with lower serum tryptophan and an elevated K/T ratio. These levels may serve as a reasonable objective marker of gut mucosal immune activation and as a surrogate for CD activity.

GATA-4, GATA-5, and GATA-6 are endodermal zinc-finger transcription factors that activate numerous enterocytic genes. GATA-4 and GATA-6 but not GATA-5 are present in adult murine small intestinal enterocytes, and we now report the simultaneous presence of all three GATA factors in murine small intestinal enterocytes before weaning age. An immunohistochemical survey detected enterocytic GATA-4 and GATA-6 at birth and 1 wk of age and GATA-5 at 1 wk but not birth. Interactions among GATA factors were explored utilizing a transgene constructed from the proximal promoter of the rat liver fatty acid binding protein gene (Fabp1). GATA-4 and GATA-5 but not GATA-6 activate the Fabp1 transgene through a cognate binding site at -128. A dose-response assay revealed a maximum in transgene activation by both factors, where additional factor did not further increase transgene activity. However, at saturated levels of GATA-4, additional transgene activation was achieved by adding GATA-5 expression construct, and vice versa. Similar cooperativity occurred with GATA-5 and GATA-6. Identical interactions were observed with a target transgene consisting of a single GATA site upstream of a minimal promoter. Furthermore, GATA-4 and GATA-5 or GATA-5 and GATA-6 bound to each other in solution. These results are consistent with tethering of one GATA factor to the Fabp1 promoter through interaction with a second GATA factor to produce increased target gene activation. Cooperative target gene activation was specific to an intestinal cell line and may represent a mechanism by which genes are activated in the small intestinal epithelium during the period before weaning.

Hepatic nuclear factor (HNF)-4 alpha and HNF-1 alpha are key endodermal transcriptional regulators that physically and functionally interact. HNF-4 alpha and HNF-1 alpha cooperatively activate genes with binding sites for both factors, whereas suppressive interactions occur at regulatory sequences with a binding site for only one factor. The liver fatty acid binding protein gene (Fabp1) has binding sites for both factors, and chromatin precipitation assays were utilized to demonstrate that HNF-4 alpha increased HNF-1 alpha Fabp1 promoter occupancy during cooperative transcriptional activation. The HNF4 P2 promoter contains a HNF-1 but not HNF-4 binding site, and HNF-4 alpha suppressed HNF-1 alpha HNF4 P2 activation and decreased promoter HNF-1 alpha occupancy. The apolipoprotein C III (APOC3) promoter contains a HNF-4 but not HNF-1 binding site, and HNF-1 alpha suppressed HNF-4 alpha APOC3 activation and decreased HNF-4 alpha promoter occupancy. Maturity onset diabetes of the young ( MODY) as well as defects in hepatic lipid metabolism result from mutations in either HNF-4 alpha or HNF-1 alpha. We found that MODY missense mutant R127W HNF-4 alpha retained wildtype individual Fabp1 activation and bound to HNF-1 alpha better than wild- type HNF-4 alpha, yet did not cooperate with HNF-1 alpha or increase HNF-1 alpha Fabp1 promoter occupancy. The R127W mutant was also defective in both suppressing HNF-1 alpha activation of HNF4 P2 and decreasing HNF-1 alpha promoter occupancy. The HNF-1 alpha R131Q MODY mutant also retained wild- type Fabp1 activation and bound to HNF-4 alpha as well as the wild type but was defective in both suppressing HNF-4 alpha APOC3 activation and decreasing HNF-4 alpha promoter occupancy. These results suggest HNF-1 alpha- HNF-4 alpha functional interactions are accomplished by regulating factor promoter occupancy and that defective factor- factor interactions may contribute to the MODY phenotype.

Previously we have reported the induction of CYP102 in Bacillus megaterium by 17beta-estradiol (E2) and 4-sec-butylphenol (4-sBP). Electrophoretic mobility shift assay analyses demonstrated that E2 and 4-sBP both cause a dose-dependent disassociation of the Bm3R1 repressor protein from its binding site on the operator sequence of the CYP102 gene. Equimolar combinations of E2 and 4-sBP demonstrated additive induction of CYP102 compared to equivalent samples of E2 and 4-sBP added alone. Two gene constructs were used in this investigation. One construct designated BMC143 contained the entire regulatory region of CYP102. The other gene construct, designated BMA45, had the "Barbie box" sequence deleted. While the induction of CYP102 by 4-sBP was much higher in the BMC 143 construct, E2 induced CYP102 in both constructs to the same extent. This difference in induction of CYP102 by these two inducers indicates that they act at different sites, either on the Bm3R1 repressor protein or on positive regulatory sites, or that they act, in part, through different mechanisms.

Cytochrome P450 102 (CYP102 or Cytochrome P450(BM)(-)(3)) is induced in Bacillus megaterium by barbiturates, perioxisome proliferators, estrogen, and nonsteroidal antiinflammatory drugs. We have previously demonstrated that a CYP102 construct (BMC 143) coupled with a luciferase reporter gene can be used to identify the inducers of CYP102. We now describe the effect of added phytochemicals on the induction of CYP102 by phenobarbital (PB) in B. megaterium. The isoflavones genistein, biochanin A, coumestrol, and equol, the green tea flavanoid epicatechin, and the fungal toxin zearalenone inhibit the induction of CYP102 by PB in a dose-dependent manner. However, the isoflavone daidzein, the phytoalexin glyceollin, and catechin, an epimer of epicatechin, failed to exhibit a similar inhibitory effect on PB-mediated CYP102 induction.

CYP102 (Cytochrome P450BM-3) is induced inBacillus megateriumby barbiturates, peroxisome proliferators, and nonsteroidal anti-inflammatory drugs. We now describe the induction of CYP102 inB. megateriumby 17β-estradiol and by 4-sec-butylphenol. These estrogens interact with the repressor protein Bm3R1, causing it to dissociate with the operator of theCYP102gene and allowing transcription to occur. We have developed a stable transfection of a construct intoB. megateriumof a truncatedCYP102gene coupled with the luciferase gene in a promoterless plasmid and have used this construct to test the induction of CYP102 by these estrogens. Estradiol demonstrated a dose-dependent induction of CYP102 which saturated at a 2-fold increase at 150 μM 4 hr post-addition. 4-sec-Butylphenol produced a dose-dependent and time-dependent induction up to 300 μM and 6 hr post-induction.